Adipocytes express a functional system for serotonin synthesis, reuptake and receptor activation.

AIMS: Serotonergic pathways in the central nervous system (CNS) are activated in the regulation of food intake and body weight. We hypothesized that adipocytes, like other cells of mesenchymal origin, possess serotonin receptors and thus could be regulated by peripherally circulating serotonin. METHODS: In vivo studies: four Sprague-Dawley rats were given daily serotonin (5HT) injections subcutaneously (s.c., 25 mg/kg) for 5 days; four controls received saline. In a long-term study, 12 rats were given serotonin s.c. for 4 months, 10 controls received saline. Body weight was registered throughout the studies, and visceral adipose tissue and plasma were collected and analysed. Adipocytes were isolated from normal rat visceral abdominal adipose tissue and analysed for the expression of serotonin receptors, the serotonin transporter (5HTT/SERT), activation of serotonin synthesis (tryptophan hydroxylase 1, Tph1) and secretion and serotonin-induced leptin regulation by RT-PCR and protein analyses. RESULTS: Hyperserotoninergic rats had significantly lower body weight (-7.4 and -6.8%) and plasma leptin levels (-44 and -38%) than controls, after both short- and long-term serotonin treatment, respectively, whereas plasma ghrelin levels were unaffected. Compared to controls, serotonin induced a 40-fold upregulation of 5HTT mRNA in visceral adipose tissue after 5 days of treatment. In vitro experiments showed that adipocytes express serotonin receptors, Tph1 and 5HTT, synthesize and secrete serotonin and that serotonin regulates leptin in mature adipocytes. CONCLUSIONS: These findings show that serotonin may regulate adipocyte function in a direct manner via the blood circulation and/or paracrine and autocrine mechanisms, and not only indirectly via the CNS as previously assumed.

Feeding behavior and body weight development: lessons from rats subjected to gastric bypass surgery or high-fat diet.

Weight loss treatments include diets, drugs, physical training, and surgery, namely bariatric or obesity surgery. The current standard for bariatric surgery is gastric bypass. There are common beliefs that gastric bypass induces body weight loss because of a reduced food intake and that high-fat diet induces overweight and obesity because of overnutrition. The principal aim of the studies on rats summarized herein was to better understand the physiological mechanisms by which gastric bypass achieves body weight loss and by which high-fat diet induces obesity. The results indicated that gastric bypass efficiently reduced body weight, particularly the fat compartment, which was unlikely to be caused by early satiety, reduced food intake or malabsorption, and that large meal size, but not overnutrition, was mainly responsible for high-fat diet-induced obesity. It was unclear whether gastric ghrelin, obestatin and/or amine in the A-like cells were involved in this context.

Feeding behavior in rats subjected to gastrectomy or gastric bypass surgery.

BACKGROUND/AIM: Gastric bypass (GB) is usually designed to restrict food intake and to induce malabsorption. Gastric hormones have been thought to play a role in the regulation of food intake and body weight. The aim of the present study was to analyze feeding behavior after total gastrectomy (Gx) or GB in rats. METHODS: Animals were subjected to Gx, GB, or sham operations. Eating and drinking behaviors after surgeries were assessed by a comprehensive laboratory animal monitoring system. Gastric hormones were measured by radioimmunoassay and energy density in feces by adiabatic bomb calorimeter. RESULTS: Compared with sham operation, both Gx and GB reduced the body weight as measured during 3-8 weeks postoperatively, which was associated with increased energy expenditure per 100 g body weight. Daily accumulated food intake and meal size (during nighttime) were reduced following Gx, but not GB. The water intake (during daytime) was increased after Gx and GB. The energy density in feces was unchanged. Serum concentrations of ghrelin, obestatin, leptin, gastrin, and pancreastatin were greatly reduced after Gx. CONCLUSIONS: Control of food intake and meal size was independent of the food reservoir function of the stomach. Surgical depletion of gastric hormones is associated with reduced meal size, but increased water intake.

Serotonin and fluoxetine modulate bone cell function in vitro.

Recent studies have proposed a role for serotonin and its transporter in regulation of bone cell function. In the present study, we examined the in vitro effects of serotonin and the serotonin transporter inhibitor fluoxetine "Prozac" on osteoblasts and osteoclasts. Human mononuclear cells were differentiated into osteoclasts in the presence of serotonin or fluoxetine. Both compounds affected the total number of differentiated osteoclasts as well as bone resorption in a bell-shaped manner. RT-PCR on the human osteoclasts demonstrated several serotonin receptors, the serotonin transporter, and the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase 1 (Tph1). Tph1 expression was also found in murine osteoblasts and osteoclasts, indicating an ability to produce serotonin. In murine pre-osteoclasts (RAW264.7), serotonin as well as fluoxetine affected proliferation and NFkappaB activity in a biphasic manner. Proliferation of human mesenchymal stem cells (MSC) and primary osteoblasts (NHO), and 5-HT2A receptor expression was enhanced by serotonin. Fluoxetine stimulated proliferation of MSC and murine preosteoblasts (MC3T3-E1) in nM concentrations, microM concentrations were inhibitory. The effect of fluoxetine seemed direct, probably through 5-HT2 receptors. Serotonin-induced proliferation of MC3T3-E1 cells was inhibited by the PKC inhibitor (GF109203) and was also markedly reduced when antagonists of the serotonin receptors 5-HT2B/C or 5-HT2A/C were added. Serotonin increased osteoprotegerin (OPG) and decreased receptor activator of NF-kappaB ligand (RANKL) secretion from osteoblasts, suggesting a role in osteoblast-induced inhibition of osteoclast differentiation, whereas fluoxetine had the opposite effect. This study further describes possible mechanisms by which serotonin and the serotonin transporter can affect bone cell function.

Immunolocalization of cholecystokinin-2 receptors in rat gastric mucosa.

BACKGROUND: Gastrin exerts trophic effects on the gastric mucosa by mechanisms not yet completely elucidated. Our aim was to localize the cholecystokinin-2 (CCK2) receptor in epithelial cells of foetal and adult rat stomachs in order to determine the cell types that are directly affected by gastrin. METHODS: Gastric tissue was subjected to indirect double immunofluorescence staining with antiserum against the C-terminal decapeptide of the CCK2 receptor and antibodies against 5' bromo-2-deoxyuridine, which had been injected into the rats I h before they were killed, the acid pump H,K-ATPase, the membrane-cytoskeletal linker ezrin, pepsin/pepsinogen or histidine decarboxylase. RESULTS: Undifferentiated foetal gastric epithelial cells expressed CCK2 receptors, whereas stem cells of adult gastric glands did not exhibit immunoreactivity. However, other epithelial cells in the progenitor zone of adult gastric glands did express CCK2 receptors. Some of these cells were faintly stained for H,K-ATPase; pepsin/pepsinogen was also detected in this region. Parietal cells in the isthmus/pit region of the glands contained ezrin, and some showed weak immunoreactivity for the CCK2 receptor. As expected, enterochromaffin-like cells also expressed CCK2 receptors. CONCLUSION: Our findings are consistent with the hypothesis that a CCK2 receptor mediates direct effects of gastrin on gastric epithelial cells during both stomach organogenesis and adult life.

Characterization of oxyntic glands isolated from the rat gastric mucosa.

A simple and reproducible method for isolating oxyntic glands from the rat gastric mucosa was developed. The mucosa was incubated with pronase and EGTA, and then treated mechanically to release glands that were separated from single cells by sedimentation. Parietal cells were identified by immunostaining using a monoclonal antibody against H,K-ATPase. The glandular cells appeared morphologically intact. By careful control of the conditions of gland isolation, long glandular structures comprising hundreds of cells surrounding the lumen were obtained. Intraperitoneal injection of Br-deoxyuridine in the rat 1.5 h before the isolation procedure resulted in glands with a labeling of cells in their neck region. The glands were viable, as demonstrated by their ability to respond to various hormones. Histamine dose-dependently stimulated the acid formation which was measured as the accumulation of [14C]aminopyrine. At 100 microM histamine the accumulation was increased 5-10-fold. At 100 nM, pentagastrin potentiated the histamine stimulated accumulation by approximately 40% but pentagastrin alone did not stimulate. The oxyntic glands obtained by the present procedure appear useful for studies on cell physiology, including regulation of acid secretion, cellular interactions, and possibly also differentiation and proliferation mechanisms since long glandular fragments that contained the proliferative zone could be isolated.

Expression of extracellular matrix proteins in the fetal rat gastric mucosa.

At gestational day 16 the epithelium of the rat stomach consists of a stratified layer of undifferentiated cells, and two days later glandular structures appear. The present study was carried out to identify extracellular matrix proteins that could be involved in the epithelial cell proliferation and differentiation processes that occur in the fetal rat stomach during this period. For comparative purposes the expression of the same components in the adult gastric mucosa was examined. Pregnant Sprague-Dawley rats received an intraperitoneal injection of 5-bromo-2'-deoxyuridine to label proliferating cells. One, 3.5, or 6 h post-injection the stomachs were excised and immediately frozen. The specimens were sectioned and stained with hematoxylin and eosin or for 5-bromo-2'-deoxyuridine, cytokeratin no. 8, H,K-ATPase, and the extracellular matrix proteins fibronectin, laminin, and collagens type I and IV. A stratified layer of proliferating cells was observed in the epithelium of the fetal stomachs, while in adult stomachs proliferating cells were detected in the isthmus/neck region of the glands. Cytokeratin, an epithelial cell marker, was sparse at gestational day 16 but abundant both at gestational day 18 and in the isthmus/neck region of gastric glands of the adult stomach. The parietal cell marker H,K-ATPase could not be detected in the fetal stomachs during this period. Fibronectin was observed in the stroma of both fetal and adult stomachs. Collagen type I could only be detected in the stroma close to the oesophagus at gestational day 16. Two days later, collagen type I was abundant in the lamina propria, the submucosa and in the serosa of the fetal stomachs. In adult tissue collagen type I was detected in the surface epithelium, the submucosa and in the serosa of the stomach. Collagen type IV and laminin were expressed in the lamina propria, the basement membranes around blood vessels, muscle cells, and nerve bundles, as well as in the serosa of both 16- and 18-day-old fetal and adult rat stomachs. In conclusion, a high cell proliferation rate was observed in the epithelium at both gestational days 16 and 18. The increased expression of cytokeratin observed during this period indicates that the epithelial character of the embryonic cells becomes more distinct, while the remarkable change in the expression of collagen type I might reflect an important role of collagen type I in the development of the gastric epithelium.

Proliferation and differentiation of cells from explants of fetal rat stomach.

The current understanding of the mechanisms controlling the proliferation and differentiation of the stem cells of the gastric oxyntic glands is limited. The aim of the present study was to develop a method for investigating proliferation and differentiation of undifferentiated cells from fetal rat stomach. Outgrowth of cells was initiated from explants of 16-day-old fetal rat stomachs. At this stage of the fetal development the gastric epithelial cells are undifferentiated. The explants were cultured in DMEM/F-12 medium supplemented with fetal calf serum only, or fetal calf serum combined with either hydrocortisone or pentagastrin. Morphological characterization by means of light microscopy, dye staining and immunostaining was used to identify the growing cells. Both hydrocortisone and pentagastrin accelerated the differentiation towards H,K-ATPase-positive cells, mucus-producing cells and other epithelial cells. H,K-ATPase-positive cells, which were identified by immunostaining with a monoclonal antibody reacting with the alpha-subunit of the H,K-ATPase, grew on top of the confluent layer of epithelioid and fibroblastoid cells. With this method in vitro investigations of the mechanisms of proliferation and differentiation of gastric mucosal cells are possible. Although by different mechanisms, both hydrocortisone and pentagastrin appear to play a regulatory role in these processes.